In vitro studies of ocular bioavailability of active pharmaceutical ingredients (API) from colloidal drug delivery systems do not consider physiological shear stress generated by eyelid wiping and ...tear flow. The present study introduces a live cell imaging approach which enables the investigation of model drug uptake from various formulations under shear stress by using custom-made microchannels for the cultivation of human corneal epithelial cells (HCE-T). Coumarin-6 (C-6) was used as a model API incorporated into solid lipid nanoparticles and liposomes, and as an aqueous crystalline suspension. Confocal laser scanning microscopy visualized C-6 uptake into HCE-T cells in a time-resolved manner with an applied shear stress of 0.1 Pa. Static conditions were also studied for comparative purposes. Additionally, solid lipid nanoparticles (SLN) were labeled with a fluorescent phospholipid to check whether C-6 uptake was associated with SLN incorporation into the cells. Results: Intact SLN were not incorporated into the cells, i.e., C-6 was passively redistributed from SLN to lipophilic cellular compartments. C-6 was enriched up to a given limit in HCE-T cells within 5 min of contact with the dispersions both under static and under flow conditions. The C-6 delivery rate from liposomes was superior to that from SLN whereby the suspension exhibited the lowest rate. C-6 release rates were comparable for static and flow conditions. Alternate flushing with formulations and buffer revealed that cells accumulated C-6. The results suggest that combining microfluidics with live cell imaging provides a valuable option for in vitro studies of ocular drug delivery.
Intermodal road-rail transport (IRRT) has a significant urban dimension, which affects the modal shift potential and the environmental benefits of rail freight. This paper explores the relevance of ...local policies for sustainable modal shift strategies by conceptualising the links between urban planning and rail freight. The presented framework identifies measures that local authorities can apply in order to increase the market and environmental improvement potential of IRRT. The results indicate that local urban transport planning has a significant role to play in the promotion of rail freight. Integrating rail freight into long-term urban development plans offers new possibilities for rail freight that are necessary in order to achieve a sustainable freight transport system in the face of ever-increasing road transport volumes.
•Urban context of rail freight affects its environmental and economic performance.•Framework to conceptualise links between urban planning and rail freight.•Local policy measures can improve rail freight performance.•Local authorities have a significant role in the promotion of rail freight.
The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αβ heterodimer, and two heterodimer isoforms (α1β1, α2β1) were characterized to have ...enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against β-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α1-, β1-, and α2-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α1-, β1-, and α2-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α1-, β1-, and α2-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.
Nitric oxide (NO) binds with high affinity to the heme of soluble guanylyl cyclase (sGC), resulting in accumulation of the second messenger cGMP in many biological systems. ...1H-1,2,4Oxadiazolo4,3-aquinoxalin-1-one (ODQ) was recently described as potent and selective inhibitor of sGC, providing an invaluable tool with which to settle the function of the cGMP pathway in NO-mediated signal transduction Mol. Pharmacol. 48:184-188 (1995). The present study investigated the mechanism of ODQ-induced inhibition of purified bovine lung sGC. The drug induced a rightward shift of the concentration-response curves recorded with two different NO donors and a reduction of maximal sGC activity, pointing to a mixed type of inhibition. The time course of NO-stimulated sGC activity determined in the presence of 0.3 microM ODQ showed that the inhibitory effect was time-dependent (half-time approximately 3 min) and virtually complete after about 10 min. The cyclase did not recover from ODQ-induced inhibition upon extensive dilution, pointing to an apparently irreversible inactivation of the enzyme by the quinoxalin. Light absorbance spectroscopy showed that ODQ (0.3 mM) induced a shift of the Soret band of the heme from 431 nm to 393 nm, indicating that ODQ oxidizes the ferrous form of the enzyme to the ferric species, which is though to exhibit only poor NO sensitivity. Together, our results suggest that inhibition of sGC by ODQ is NO-competitive and results in an apparently irreversible oxidation of the prosthetic heme group.
The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αβ heterodimer, and two heterodimer isoforms (α
1
β
1
, α
2
β
1
) were characterized to ...have enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against β-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α
1
-, β
1
-, and α
2
-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α
1
-, β
1
-, and α
2
-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α
1
-, β
1
-, and α
2
-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.
By the formation of cyclic guanosine 3′,5′-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, ...which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC α2-subunit/β1-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC α2, and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10−5 M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10−4 M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.
The demand for inland freight transport in Europe is mainly met by road transport, leading to unsustainable impacts such as air pollution, greenhouse gas emissions and congestion. Since rail ...transport has lower externalities than road transport, a modal shift from road to rail is an accepted policy goal for achieving a more sustainable and competitive transport system. However, intermodal road-rail transport is mainly competitive for long-distance transport, and as a consequence, the potential for modal shift is limited. The cost efficiency of road-rail intermodal transport is particularly sensitive to pre- and post-haulage (PPH) costs, since this activity typically has a larger cost compared with its share of the total distance in the transport chain. For intermodal transportation over shorter distances, for example, below 300 km and where there are substantial PPH activities at both ends of the chain, the competitiveness of the intermodal transport system compared with that of direct road is low. Improving the efficiency of PPH activities is, therefore, of utmost importance for the competitiveness of the intermodal transport system. This paper looks into the issue of improving the cost efficiency of an intermodal transport chain by implementing an innovative and flexible legal framework regarding the PPH activities in the chain. By extending the legal framework with exemptions for longer vehicles in PPH, the cost efficiency could be greatly improved. The purpose of such a framework is to allow and enable, for PPH exclusively, the use of 2 × 40 foot or even two semi-trailers using only one vehicle in the context of the Swedish regulatory framework. This paper develops a strategic calculation model for assessing and investigating the consequences of such a framework and investigates the framework's potential in terms of cost efficiency. The model in combination with a sensitivity analysis of input variables gives a comprehensive understanding of the effects of PPH under different circumstances. From the results, it is evident that there are substantial positive effects associated with a PPH framework of longer vehicles. Results indicate that a typical shipper may experience cost reductions of about 5-10% of the total costs of the intermodal transport chain. In summary, a more innovative and flexible legal framework regarding vehicle length in the PPH links can contribute to a greater modal shift, improved cost efficiency and more environmentally friendly transportation systems.
The messenger molecule cyclic guanosine monophosphate (cGMP) is produced by different isoforms of the enzyme guanylate cyclase (GC). Natriuretic peptides (ANP and CNP) bind to and activate ...particulate GCs, whereas NO and CO activate a soluble form of GC. The specific relevance of the cGMP system for reproductive functions has been recently demonstrated by the successful use of sildenafil (Viagra), an inhibitor of cGMP-specific phosphodiesterase type 5, for the treatment of erectile dysfunction. In the testis, cGMP signal transduction pathways are involved in a variety of local functions, based on autocrine or paracrine effects. In particular, cGMP has been suggested to influence motility in spermatozoa, development of testicular germ cells, relaxation of peritubular lamina propria cells, testosterone synthesis in Leydig cells and dilatation of testicular blood vessels. The physiological significance of cGMP accumulation in Scrtoli cells is not yet clear. Taken as a whole, the evidence suggests that cGMP-mediated processes might influence both the potentia coeundi within the penis and the potentia generandi at various levels within the testis.
Soluble guanylyl cyclase (sGC), the target enzyme of the signalling molecule NO, contains one prosthetic haem group and consists of an alpha and a beta subunit. So far, only the alpha1beta1 ...heterodimer has been shown to exist in different cells and tissues, and most biochemical studies of sGC have been performed with the alpha1 beta1 heterodimer. Here we demonstrate for the first time the natural occurrence of the alpha2 subunit on the protein level. The alpha2 subunit co-precipitated with the beta1 subunit from human placenta, showing the existence of the alpha2 beta1 isoform in vivo. The new enzyme was expressed in and purified from cells from the Spodoptera frugiperda ovary cell line Sf 9. Spectral analysis showed that the alpha2 beta1 heterodimer contains a prosthetic haem group revealing the same characteristics as the haem in the alpha1 beta1 form. The kinetic properties of both isoforms and sensitivity towards NO were indistinguishable. 1H-1,2,4oxadiazolo4,3-aquinoxalin-1-one (ODQ), a selective inhibitor of sGC, abolished NO-stimulated activity of both heterodimers. The new NO-independent activator, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), increased the maximal NO-stimulated activity of the new isoform, caused a leftward-shift in the NO concentration-response curve and turned CO into an effective activator, as it did for the alpha1 beta1 heterodimer (200-fold activation). In summary, the differences in primary structure of both alpha subunits are contrasted by their functional similarity. Further studies will be needed to elucidate the physiological purpose of the new isoform.
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